Production of proinflammatory cytokines by macrophages significantly contributes to the pathogenesis of many inflammatory diseases including arthritis. Therefore, understanding the signaling mechanisms involved in the regulation of expression of pro-inflammatory molecules by macrophages is pivotal to developing drugs to combat inflammatory diseases. Recent studies have implicated Toll-like receptors (TLRs) in the initiation and maintenance of inflammation in a number of human diseases. Of particular importance is the role of TLR4, which critically regulates cytokine production and is therefore a drug target in inflammatory diseases. Hence, understanding the biochemical mechanisms by which TLR4-stimulated signaling pathways affect expression of inflammatory molecules is highly significant. Recent studies have demonstrated a critical role for non-visual arrestins (ARR2 and ARR3) in the regulation of TLR signaling and pathophysiology. Arrestins (ARRs) are scaffolding proteins originally discovered for their role in G- protein coupled receptor (GPCR) desensitization. Evidence indicates that ARR2 and 3 interact with distinct proteins and consequently regulate TLR4 signaling and inflammatory gene expression in macrophages and in vivo in mice. The objective of this application is to understand the biochemical mechanisms by which ARR2 and ARR3 regulate TLR4 signaling in macrophages and to test how this relates to inflammation in mice. To accomplish this objective, we will test the following hypotheses: (a) ARR2 and 3 play a crucial role in TLR4 signaling, and inflammatory mediator production in macrophages, and (b), this occurs via distinct protein-protein interactions, and (c) ARR2 and 3 are critically involved in TLR4-mediated inflammatory response in mice in vivo. Our studies should provide important insight into the mechanisms of TLR4 signaling in macrophages as well as provide potential therapeutic strategies for targeting TLR4 signaling in a variety of inflammatory diseases. PUBLIC HEALTH RELEVANCE: Toll-like receptor-4 activation in macrophages plays an important role in the development of various inflammatory diseases. Major objective of this application is to understand the regulation of TLR4-stimulated signaling pathways in macrophages and test as to how it relates to inflammatory diseases.